"Do it yourself" protocol to fabricate dual-detection paper-based analytical device for salivary biomarker analysis.

This paper describes the design and construction of dual microfluidic paper-based analytical devices (dual-µPADs) as a lab-on-paper platform involving a "do-it-yourself" fabrication protocol. The device comprises a colorimetric and electrochemical module to obtain a dual-mode signal readout sensing strategy. A 3D pen polymeric resin was used to prepare graphite carbon-based electrodes and hydrophobic barriers on paper substrates. The proposed carbon-based ink was employed to manufacture electrodes on paper based on a stencil-printing approach, which were further characterized by electrochemical and morphological analyses. The analytical performance of the dual-µPADs was simultaneously evaluated for lactate, pH, nitrite, and salivary amylase (sAA) analysis. To demonstrate the proof-of-concept, saliva samples collected from both healthy individuals and those with periodontitis were successfully tested to demonstrate the feasibility of the proposed devices. Samples collected from individuals previously diagnosed with periodontitis showed high levels of nitrite and sAA (> 94 µmol L-1 and > 610 U mL-1) in comparison with healthy individuals (≤ 16 µmol L-1 and 545 U mL-1). Moreover, periodontitis saliva resulted in acid solution and almost null lactate levels. Notably, this protocol supplies a simple way to manufacture dual-µPADs, a versatile platform for sensitive detecting of biomarkers in saliva playing a crucial role towards the point-of-care diagnosis of periodontal disease.

Nondestructive, reagent-free, low-volume fluidic set-up to study biofilms by using a transparent electrode, allowing simultaneous electrochemical and optical measurements.

AIMS: The aim was to develop an electrochemical/optical set-up and correlate it (as validation) with other chemical and physical methods to obtain a simple and cost-effective system to study biofilm formation. METHODS AND RESULTS: A simple microfluidic cell and methods allowed continuous monitoring of the first, critical steps of microbial attachment. We monitored sulfate-reducing bacteria (SRB) at the early stages of biofilm formation. Herein, we studied the formation and adherence of SRB consortium biofilms over an indium tin oxide (ITO) conducting surface using microbiological and chemical methods, microscopic observations [scanning electron microscopy (SEM) and optical], and electrochemical impedance spectroscopy (EIS) measurements. The SRB biofilm formation was evaluated for 30 d by SEM and EIS. Charge transfer resistance decreased when the microbial population colonized the electrode. The monitoring of early-stage biofilm formation was performed using EIS at a single frequency of 1 Hz during the first 36 h. CONCLUSIONS: The simultaneous use of optical, analytical, and microbiological methods allowed us to connect the kinetics of the growth of the microbial consortium to the values obtained via the electrochemical technique. The simple setup we present here can help laboratories with limited resources to study biofilm attachment and facilitates the development of various strategies to control biofilm development in order to avoid damage to metallic structures (microbiologically influenced corrosion, MIC) or the colonization of other industrial structures and medical devices.

Phytoremediation of toxic chemicals in aquatic environment with special emphasis on duckweed mediated approaches.

The discharge of toxic chemicals into water bodies and their linked detrimental effects on health is a global concern. Phytoremediation, an environment-friendly plant-based technology, has gained intensive interest over the last decades. For the aquatic phytoremediation process, the commonly available duckweeds have recently attracted significant attention due to their capacity to grow in diverse ecological niches, fast growth characteristics, suitable morphology for easy handling of biomass, and capacity to remove and detoxify various potential toxic elements and compounds. This review presents the progress of duckweed-assisted aquatic phytoremediation of toxic chemicals. A brief background of general phytoremediation processes, including the different phytoremediation methods and advances in understanding their underlying mechanisms, has been described. A summary of different approaches commonly practiced to assess the growth of the plants and their metal removal capacity in the phytoremediation process has also been included. A vast majority of studies have established that duckweed is an efficient plant catalyst to accumulate toxic heavy metals and organic contaminants, such as pesticides, fluorides, toxins, and aromatic compounds, reducing their toxicity from water bodies. The potential of this plant-based phytoremediation process for its downstream applications in generating value-added products for the rural economy and industrial interest has been identified.

Duckweed is an aquatic plant widely available in diverse ecosystems on the earth. Due to its fast growth in various environmental conditions, capacity to accumulate and transform different toxic chemicals, and a suitable morphology for handling and processing its biomass easily, duckweed has been projected as an efficient floating plant species for the aquatic phytoremediation technology. Moreover, the duckweed biomass generated from the post phytoremediation process may be transformed into various value-added products to support the rural economy.

A new P. putida instrumental toxicity bioassay.

Here, we present a new toxicity bioassay (CO2-TOX), able to detect toxic or inhibitory compounds in water samples, based on the quantification of Pseudomonas putida KT2440 CO2 production. The metabolically produced CO2 was measured continuously and directly in the liquid assay media, with a potentiometric gas electrode. The optimization studies were performed using as a model toxicant 3,5-DCP (3,5-dichlorophenol); later, heavy metals (Pb(2+), Cu(2+), or Zn(2+)) and a metalloid (As(5+)) were assayed. The response to toxics was evident after 15 min of incubation and at relatively low concentrations (e.g., 1.1 mg/L of 3,5-DCP), showing that the CO2-TOX bioassay is fast and sensitive. The EC50 values obtained were 4.93, 0.12, 6.05, 32.17, and 37.81 mg/L for 3,5-DCP, Cu(2+), Zn(2+), As(5+), and Pb(2+), respectively, at neutral pH. Additionally, the effect of the pH of the sample and the use of lyophilized bacteria were also analyzed showing that the bioassay can be implemented in different conditions. Moreover, highly turbid samples and samples with very low oxygen levels were measured successfully with the new instrumental bioassay described here. Finally, simulated samples containing 3,5-DCP or a heavy metal mixture were tested using the proposed bioassay and a standard ISO bioassay, showing that our test is more sensible to the phenol but less sensible to the metal mixtures. Therefore, we propose CO2-TOX as a rapid, sensitive, low-cost, and robust instrumental bioassay that could perform as an industrial wastewater-process monitor among other applications.

Mixture toxicity study of two metal oxide nanoparticles and chlorpyrifos on Eisenia andrei earthworms.

Co-exposure soil studies of pollutants are necessary for an appropriate ecological risk assessment. Here, we examined the effects of two-component mixtures of metal oxide nanoparticles (ZnO NPs or goethite NPs) with the insecticide chlorpyrifos (CPF) under laboratory conditions in short-term artificial soil assays using Eisenia andrei earthworms. We characterized NPs and their mixtures by scanning electron microscopy, atomic force microscopy, dynamic light scattering and zeta potential, and evaluated effects on metal accumulation, oxidative stress enzymes, and neurotoxicity related biomarkers in single and combined toxicity assays. Exposure to ZnO NPs increased Zn levels compared to control in single and combined exposure (ZnO NPs + CPF) at 72 h and 7 days, respectively. In contrast, there was no indication of Fe increase in organisms exposed to goethite NPs. One of the most notable effects on oxidative stress biomarkers was produced by single exposure to goethite NPs, showing that the worms were more sensitive to goethite NPs than to ZnO NPs. Acetylcholinesterase and carboxylesterase activities indicated that ZnO NPs alone were not neurotoxic to earthworms, but similar degrees of inhibition were observed after single CPF and ZnO NPs + CPF exposure. Differences between single and combined exposure were found for catalase and superoxide dismutase (goethite NPs) and for glutathione S-transferase (ZnO NPs) activities, mostly at 72 h. These findings suggest a necessity to evaluate mixtures of NPs with co-existing contaminants in soil, and that the nature of metal oxide NPs and exposure time are relevant factors to be considered when assessing combined toxicity, as it may have an impact on ecotoxicological risk assessment.

A review of the recent achievements and future trends on 3D printed microfluidic devices for bioanalytical applications.

3D printing has revolutionized the manufacturing process of microanalytical devices by enabling the automated production of customized objects. This technology promises to become a fundamental tool, accelerating investigations in critical areas of health, food, and environmental sciences. This microfabrication technology can be easily disseminated among users to produce further and provide analytical data to an interconnected network towards the Internet of Things, as 3D printers enable automated, reproducible, low-cost, and easy fabrication of microanalytical devices in a single step. New functional materials are being investigated for one-step fabrication of highly complex 3D printed parts using photocurable resins. However, they are not yet widely used to fabricate microfluidic devices. This is likely the critical step towards easy and automated fabrication of sophisticated, complex, and functional 3D-printed microchips. Accordingly, this review covers recent advances in the development of 3D-printed microfluidic devices for point-of-care (POC) or bioanalytical applications such as nucleic acid amplification assays, immunoassays, cell and biomarker analysis and organs-on-a-chip. Finally, we discuss the future implications of this technology and highlight the challenges in researching and developing appropriate materials and manufacturing techniques to enable the production of 3D-printed microfluidic analytical devices in a single step.

Improved sensitivity in paper-based microfluidic analytical devices using a pH-responsive valve for nitrate analysis.

This paper presents an innovative application of chitosan material to be used as pH-responsive valves for the precise control of lateral flow in microfluidic paper-based analytical devices (µPADs). The fabrication of µPADs involved wax printing, while pH-responsive valves were created using a solution of chitosan in acetic acid. The valve-forming solution was applied, and ready when dry; by exposure to acidic solutions, the valve opens. Remarkably, the valves exhibited excellent compatibility with alkaline, neutral, and acidic solutions with a pH higher than 4. The valve opening process had no impact on the flow rate and colorimetric analysis. The potential of chitosan valves used for flow control was demonstrated for µPADs employed for nitrate determination. Valves were used to increase the conversion time of nitrate to nitrite, which was further analyzed using the Griess reaction. The µPAD showed a linear response in the concentration range of 10-100 µmol L-1, with a detection limit of 5.4 µmol L-1. As a proof of concept, the assay was successfully applied to detect nitrate levels in water samples from artificial lakes of recreational parks. For analyses that require controlled kinetics and involve multiple sequential steps, the use of chitosan pH-responsive valves in µPADs is extremely valuable. This breakthrough holds great potential for the development of simple and high-impact microfluidic platforms that can cater to a wide range of analytical chemistry applications.

A laboratory experiment for science courses: Sedimentary microbial fuel cells.

Nowadays there is a concern to improve the quality of education by including an interdisciplinary approach of concepts and their integration in the curriculum of scientific disciplines. The development of microbial fuel cells as a potential alternative for production of renewable energies gives undergraduate students the challenge of integrating interdisciplinary concepts in a hot topic of global interest as alternative energies. We present a laboratory experiment that has been part of a third-year undergraduate course in biology where students gained experience in assembling microbial fuel cells and the understanding of how they work. In this process, the students could integrate biological, biochemical, and electric concepts. In addition, the acquisition of manual skills and experimental design decisions are important for the development of future professionals.

When microplastics meet electroanalysis: future analytical trends for an emerging threat.

Microplastics are a major modern challenge that must be addressed to protect the environment, particularly the marine environment. Microplastics, defined as particles ≤5 mm, are ubiquitous in the environment. Their small size for a relatively large surface area, high persistence and easy distribution in water, soil and air require the development of new analytical methods to monitor their presence. At present, the availability of analytical techniques that are easy to use, automated, inexpensive and based on new approaches to improve detection remains an open challenge. This review aims to outline the evolution and novelties of classical and advanced methods, in particular the recently reported electroanalytical detectors, methods and devices. Among all the studies reviewed here, we highlight the great advantages of electroanalytical tools over spectroscopic and thermal analysis, especially for the rapid and accurate detection of microplastics in the sub-micron range. Finally, the challenges faced in the development of automated analytical methods are discussed, highlighting recent trends in artificial intelligence (AI) in microplastics analysis.

Voltamperometric discrimination of urea and melamine adulterated skimmed milk powder.

Nitrogen compounds like urea and melamine are known to be commonly used for milk adulteration resulting in undesired intoxication; a well-known example is the Chinese episode occurred in 2008. The development of a rapid, reliable and economic test is of relevance in order to improve adulterated milk identification. Cyclic voltammetry studies using an Au working electrode were performed on adulterated and non-adulterated milk samples from different independent manufacturers. Voltammetric data and their first derivative were subjected to functional principal component analysis (f-PCA) and correctly classified by the KNN classifier. The adulterated and non-adulterated milk samples showed significant differences. Best results of prediction were obtained with first derivative data. Detection limits in milk samples adulterated with 1% of its total nitrogen derived from melamine or urea were as low as 85.0 mg · L(-1) and 121.4 mg · L(-1), respectively. We present this method as a fast and robust screening method for milk adulteration analysis and prevention of food intoxication.

Archaea-based microbial fuel cell operating at high ionic strength conditions.

In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC's performance. Without any added redox mediator, maximum power (P (max)) and current at P (max) were 11.87/4.57/0.12 µW cm(-2) and 49.67/22.03/0.59 µA cm(-2) for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P (max) was 50.98 and 5.39 µW cm(-2) for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.

Sorting the main bottlenecks to use paper-based microbial fuel cells as convenient and practical analytical devices for environmental toxicity testing.

Three of the primary bottlenecks, which should be consider for practical, point-of-need use of microbial fuel cell (MFC) analytical devices were surpassed in this work: i) the use of a diffusive barrier, hence, an electrogenic biofilm; ii) longer enrichment/stabilization times to produce a biofilm, made in a laboratory environment, over the electrode; and iii) difficulty comparing results obtained from MFCs based on electrogenic biofilms with standardized bioassays, a setback to be adopted as a new method. Here we show an easy way to determine water toxicity employing planktonic bacteria as biorecognition agents. The paper-based MFC contain an electron carrier (or mediator) to facilitate charge transfer from bacteria to the anode. In this way, there is no need to use biofilms. As far as we know this is the first paper-based MFC containing P. putida KT2440, a well characterized non-pathogenic bacteria previously used in standardized water toxicity bioassays. Results were obtained in 80 min and an effective concentration 50 of 9.02 mg L-1, calculated for Zn2+ (a reference toxic agent), was successfully compared with previously published and ISO standardized bioassays, showing a promising future for this technology. The practical design and cost (less than one U.S. dollar) of the paper-based MFC toxicity test presented will open new market possibilities for rapid and easy-to-use MFC analytical devices.

Hydrogen production in two-chamber MEC using a low-cost and biodegradable poly(vinyl) alcohol/chitosan membrane.

Hydrogen production was evaluated in two-chamber microbial electrolysis cells (MEC), where the chambers of the cell were separated using a new economical and environmentally friendly membrane made of poly (vinyl) alcohol/chitosan (PVA/CS). The MEC performance was compared to that of Nafion. The obtained results indicated that the MEC performance for hydrogen production did not show significant differences between the PVA/CS and Nafion membranes. MEC with PVA/CS showed the hydrogen production rate and hydrogen yield of 1277 ± 46 mL H2Lcat-1d-1 and 974 ± 116 mL H2 gacetate-1, respectively. The PVA/CS membrane allowed acetate removal that was 7% higher than that of Nafion due to the lower pH gradient and a lower voltage drop that increased the ion transfer across the membrane.

An apta-aggregation based machine learning assay for rapid quantification of lysozyme through texture parameters.

A novel assay technique that involves quantification of lysozyme (Lys) through machine learning is put forward here. This article reports the tendency of the well- documented Ellington group anti-Lys aptamer, to produce aggregates when exposed to Lys. This property of apta-aggregation has been exploited here to develop an assay that quantifies the Lys using texture and area parameters from a photograph of the elliptical aggregate mass through machine learning. Two assay sets were made for the experimental procedure: one with high Lys concentration between 25-100 mM and another with low concentration between 1-20 mM. The high concentration set had a sample volume of 10 µl while the low concentration set had a higher sample volume of 100 µl, in order to obtain the statistical texture values reliably from the aggregate mass. The platform exhibited an experimental limit of detection of 1 mM and a response time of less than 10 seconds. Further, two potential operating modes for the aptamer were hypothesized for this aggregation property and the more accurate mode among the two was ascertained through bioinformatics studies.

Towards a versatile and economic Chagas Disease point-of-care testing system, by integrating loop-mediated isothermal amplification and contactless/label-free conductivity detection.

Rapid diagnosis by using small, simple, and portable devices could represent one of the best strategies to limit the damage and contain the spread of viral, bacterial or protozoa diseases, principally when they can be transmitted by air and are highly contagious, as some respiratory viruses are. The presence of antibodies in blood or serum samples is not the best option for deciding when a person must be quarantined to stop transmission of disease, given that cured patients have antibodies, so the best diagnosis methods rely on the use of nucleic acid amplification procedures. Here we present a very simple device and detection principle, based on paper discs coupled to contactless conductivity (C4D) sensors, can provide fast and easy diagnostics that are needed when an epidemic outbreak develops. The paper device presented here solves one of the main drawbacks that nucleic acid amplification tests have when they are performed outside of central laboratories. As the device is sealed before amplification and integrally disposed in this way, amplimers release cannot occur, allowing repetitive testing in the physician's practice, ambulances, or other places that are not prepared to avoid cross-contamination of new samples. The use of very low volume samples allows efficient reagent use and the development of low cost, simple, and disposable point-of-care diagnostic systems.

Development of a Novel Method for in vivo Determination of Activation Energy of Glucose Transport Across S. cerevisiae Cellular Membranes. A Biosensor-like Approach.

Whereas biosensors have been usually proposed as analytical tools, used to investigate the surrounding media pursuing an analytical answer, we have used a biosensor-like device to characterize the microbial cells immobilized on it. We have studied the kinetics of transport and degradation of glucose at different concentrations and temperatures. When glucose concentrations of 15 and 1.5 mM were assayed, calculated activation energies were 25.2 and 18.4 kcal mol(-1), respectively, in good agreement with previously published data. The opportunity and convenience of using Arrhenius plots to estimate the activation energy in metabolic-related processes is also discussed.

Characterization of a new composite membrane for point of need paper-based micro-scale microbial fuel cell analytical devices.

Microbial fuel cells (MFCs) can evolve in a viable technology if environmentally sound materials are developed and became available at low cost for these devices. This is especially important not only for the designing of large wastewater treatment systems, but also for the fabrication of low-cost, single-use devices. In this work we synthesized membranes by a simple procedure involving easily-biodegradable and economic materials such as poly (vinyl alcohol) (PVA), chitosan (CS) and the composite PVA:CS. Membranes were chemical and physically characterized and compared to Nafion®. Performance was studied using the membrane as separator in a typical H-Type MFCs showing that PVA:CS membrane outperform Nafion® 4 times (power production) while being 75 times more economic. We found that performance in MFC depends over interactions among several membrane characteristics such as oxygen permeability and ion conductivity. Moreover, we design a paper-based micro-scale MFC, which was used as a toxicity assay using 16 µL samples containing formaldehyde as a model toxicant. The PVA:CS membrane presented here can offer low environmental impact and become a very interesting option for point of need single-use analytical devices, especially in low-income countries where burning is used as disposal method, and toxic fluoride fumes (from Nafion®) can be released to the environment.

Label-free counting of Escherichia coli cells in nanoliter droplets using 3D printed microfluidic devices with integrated contactless conductivity detection.

This study describes for the first time the development of 3D printed microfluidic devices with integrated electrodes for label-free counting of E. coli cells incorporated inside droplets based on capacitively coupled contactless conductivity detection (C4D). Microfluidic devices were fully fabricated by 3D printing in the T-junction shape containing two channels for disperse and continuous phases and two sensing electrodes for C4D measurements. The disperse phase containing E. coli K12 cells and the continuous phase containing oil and 1% Span® 80 were pumped through flow rates fixed at 5 and 60 µL min-1, respectively. The droplets with incorporated cells were monitored in the C4D system applying a 500-kHz sinusoidal wave with 1 Vpp amplitude. The generated droplets exhibited a spherical shape with average diameter of 321 ±â€¯9 µm and presented volume of 17.3 ±â€¯0.5 nL. The proposed approach demonstrated ability to detect E. coli cells in the concentration range between 86.5 and 8650 CFU droplet-1. The number of cells per droplet was quantified through the plate counting method and revealed a good agreement with the Poisson distribution. The limit of detection achieved for counting E. coli cells was 63.66 CFU droplet-1. The label-free counting method has offered instrumental simplicity, low cost, high sensitivity and compatibility to be integrated on single microfluidic platforms entirely fabricated by 3D printing, thus opening new possibilities of applications in microbiology.

An electrochemical sensing approach for scouting microbial chemolithotrophic metabolisms.

The present study was aimed to test an electrochemical sensing approach for the detection of an active chemolithotrophic metabolism (and therefore the presence of chemolithotrophic microorganisms) by using the corrosion of pyrite by Acidithiobacillus ferrooxidans as a model. Different electrochemical techniques were combined with adhesion studies and scanning electron microscopy (SEM). The experiments were performed in presence or absence of A. ferrooxidans and without or with ferrous iron in the culture medium (0 and 0.5 g L-1, respectively). Electrochemical parameters were in agreement with voltammetric studies and SEM showing that it is possible to distinguish between an abiotically-induced corrosion process (AIC) and a microbiologically-induced corrosion process (MIC). The results show that our approach not only allows the detection of chemolithotrophic activity of A. ferrooxidans but also can characterize the corrosion process. This may have different kind of applications, from those related to biomining to life searching missions in other planetary bodies.

Membrane entrapped Saccharomyces cerevisiae in a biosensor-like device as a generic rapid method to study cellular metabolism.

We describe a new, faster and convenient method to study some metabolic characteristics - by the successful application of immobilized yeast cells (S. cerevisiae) in a microbial biosensor-like device. Microbial biosensors consist of microorganisms immobilized on the surface of a membrane or in a gel, in close contact with a transducer. Almost all works published to date have used biosensors for analyses in which a concentration-related property of the external medium is measured. A different approach is presented here; we have successfully used S. cerevisiae and a carbon dioxide electrode as the main components of a biosensor-like device, used as a proof of concept, for a system useful to characterize metabolic parameters of the microbial cells immobilized on a carbon dioxide electrode. The biosensor-like device we are presenting allows us to calculate Michaelis-Menten parameters related to the kinetics of transport and degradation of several carbohydrates (i.e., glucose, fructose, galactose, sucrose and xylose, with K(m(app)) of 6.0, 5.8, 0.9, 2.0, and 147 mM, respectively), and the study of the kinetics of expression of non-constitutive proteins related to the transport and degradation of galactose.